...transcribed to make the pre-CRISPR RNA (pre-crRNA). (2) The pre-crRNA is processed into individual crRNAs...2015. Class 1 (Multi-subunit effector complex) Class 2 (Single multi-domain effector) Type I (Cas3) Type ... with unprecedented speed and specificity. Figure 2: An overview of CRISPR and NHEJ/HDR. The Cas9/gRNA...regulation of transcription in eukaryotes. Cell . 154(2):442-51. PMID: 23849981 Ishino Y, Shinagawa H, Makino...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell . 163(3):759-71. PMID: 26422227... The first base editors converted cytidine to thymidine; newly engineering base editors convert adenosine...

...8661 p4455 FLAG-hPLIC-2 UBQLN2 Flag CMV ALS Peter Howley 8662 p4456 FLAG-hPLIC-2 NTF UBQLN2 Flag CMV ALS...196208 GB1-A11(2-196)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196209 GB1-A11(2-196, G38R)-Strep...196210 GB1-A11(2-196, D40G)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196211 GB1-A11(2-196, G175R...196212 GB1-A11(2-196, G189E)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196213 GB1-A11(2-52)-Strep ...TLS 1: hTLS.pBSKS(+) FUS T7 ALS David Ron 21828 TLS 2: hTLS.pCDNA1 FUS CMV ALS David Ron 21829 TLS 3: TLS.pRSET.C...ApoE Alzheimer's Sohail Tavazoie 51436 pGL3Basic-ME.2/ApoEpromoter APOE ApoE Alzheimer's Sohail Tavazoie...T7 ALS Nicolas Fawzi 127195 RP1B FUS 1-163 QQ4xSS #2 FUS His T7 ALS Nicolas Fawzi 127196 pJ411 FUS 1-163...

.... Diagnostic restriction digests are comprised of 2 separate steps: 1) incubating your DNA with restriction...which cleave the DNA molecules at specific sites and 2) running the reaction on an agarose gel to determine...including a 1.2 kb insert. The plasmid was digested with 2 unique enzymes (HindIII and BamHI) and run on an agarose...to serve as a reference band. The uncut DNA (lane 2) shows 3 possible plasmid conformations, with relaxed...Watch out for methylation issues. Enzymes like XbaI and ClaI are sensitive to methylation and their activity...will need to purify your DNA from a dcm or dam methylation-deficient bacterial strain such as JM110 or INV110...

...//doi.org/10.1002/1096-8628(200022)97:2<147::aid-ajmg6>3.0.co;2-o Baker SA, Chen L, Wilkins AD, Yu P, ...Syndrome? Caused by a mutation in the methyl-CpG binding protein 2 (MECP2) gene, Rett Syndrome is a rare...mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet 23:185–188 . https://doi.... RE, Zoghbi HY (2000) Rett syndrome: Methyl-CpG-binding protein 2 mutations and phenotype-genotype correlations...et al., 1999). We now know that MECP2 binds to methylated CpG dinucleotides and CAC trinucleotides during...

...of conventional gene targeting is lowered to about 2-3 months. But certain models remain more difficult...included in the final ssDNA template. Figure 2: Overview of ivTRT to generate an ssDNA repair template... floxed conditional allele requires you to target 2 loxP sites to intronic regions 0.5 - 0.8 kb apart....Typically, Easi-CRISPR works best with insertions of 2 kb or less. Luckily, most of the commonly used cassettes... induction systems (ttA or rtTA) are between 1 to 2 kb in length. There are plenty of Addgene plasmid ...create the ssDNA construct as described in Figure 2.  You can also use Addgene plasmids to purify Cas9...directed repair. Using long ssDNA donors, the Gurumurthy and Ohtsuka groups have obtained an average knock-in...

... to the lysosome where they are finally degraded (2, 3). Once you've created your fusion protein, it's...examples include the ImageJ plugins JACoP and Coloc 2, BioImageXD, and customized CellProfiler pipelines...colocalization analysis example is given in Figure 2 (adapted from 4) - Dunn et al. 2011 provides an in-depth...Since colocalization relies upon the detection of 2 independent fluorescent signals and their potential...here! References 1. Allen Institute Plasmid Pages 2. Cooper GM. 2000. The Cell: A Molecular Approach. ... in protein localization and transport between healthy and diseased states can also provide interesting... the function of your protein of interest in a healthy or diseased cell. We hope you find the resources...

...repository of tools for SAR-CoV-2 research. Two new antibodies, Anti-SARS-CoV-2 Nucleocapsid Protein [mBG17...Anti-SARS-CoV-2 Nucleocapsid Protein [mBG86], target distinct epitopes on the SARS-CoV-2 nucelocapsid ...Anti-SARS-CoV-2 Antibodies here! Terry, J.S., et al. (2021). Development of a SARS-CoV-2 nucleocapsid ...prime editor PE7 Antibodies to facilitate SARS-CoV-2 research New viral vector preps of Voltron2 voltage... incorporates La’s N-terminal domain, PE7 (Figure 2).  PE7 shows enhanced editing efficiencies in a number...editor. Find PE7 plasmids here!     Figure 2: PE7 improves prime editing. A) Schematic of PE7 prime...PMID: 38570691. Antibodies to facilitate SARS-CoV-2 research   By Ashley Waldron Ready-to-use recombinant...

...Citorik Plasmids here). In both cases, they used 1-2 CRISPR crRNAs targeting sequences found only in certain...resensitize antibiotic-resistant bacteria (Figure 2). The first phage introduced is lysogenic, integrating...machinery targeting 1) the given resistance gene and 2) a second lytic phage. Bacteria that receive this ...bacteria have previously flourished.   Figure 2: Two-phage strategies to resensitize bacteria to antibiotics...237163. Find plasmids from this paper at Addgene. 2. Bikard, David, et al. “Exploiting CRISPR-Cas nucleases...References 1. Citorik, Robert J., Mimee, Mark, and Timothy K. Lu. “Sequence-specific antimicrobials using ... Hiroki, Sebastien Lemire, Diana P. Pires, and Timothy K. Lu. “Engineering Modular Viral Scaffolds for...

...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors of...for generating iPSCs by plasmid transfection where 2 of 11 clones tested had plasmid integration. Another...understanding the mechanisms of reprogramming, and 2) studies with clinical endpoints. In the first scenario...PubMed PMID: 27764162. PubMed Central PMCID: 5072705. 2. Sommer, Cesar A., and Gustavo Mostoslavsky. "The ...challenges." Journal of Cellular Physiology 228, no. 2 (2012): 267-75. PubMed PMID: 22767332.  3. Kim, Dohoon...PubMed PMID: 23546745. PubMed Central PMCID: 4176696. 2. Yusa, Kosuke, Roland Rad, Junji Takeda, and Allan...generation. Expert opinion on biological therapy, 10 2, 231-42. PubMed PMID: 20088717. PubMed Central PMCID...

...Cre-ERT2 CAG Mammalian Heller 112622 pVHC_PGKneoLox2DTA.2 Venus, Cre-ERT2, targeting vector with MCS for homology...homology arms Mammalian Heller 112623 pEHC_PGKneoLox2DTA.2 Emerald, Cre-ERT2, targeting vector with MCS for homology... arms Mammalian Heller 112624 pmTHC_PGKneoLox2DTA.2 TFP, Cre-ERT2, targeting vector with MCS for homology...arms Mammalian Heller 112625 ptdTHC_PGKneoLox2DTA.2 tdTomato, Cre-ERT2, targeting vector with MCS for ...25;1504(4):467-86. doi:10.1016/0022-2836(81)90375-2. PubMed . Ventura A, Meissner A, Jaenisch R, Jacks...Cre CAG Mammalian Pelczar 51276 Thy1.2-Roxed-Cre Dre-respondent Cre Thy1 Mammalian Pelczar 51507 AAV pmSyn1...

...host range plasmids. FEMS Microbiology Letters, 348(2), 87–96. https://doi.org/10.1111/1574-6968.12241  ...mobilization of broad host-range IncQ plasmids. Plasmid, 62(2), 57–70. https://doi.org/10.1016/j.plasmid.2009.05.001...negative hosts. Biotechnology and Bioengineering, 106(2), 326–332. https://doi.org/10.1002/bit.22695  Schmidhauser...Legionella pneumophila, Methylophilus methyltrophus, Methylococcus methanolicus, Methylosinus trichosporium, Myxococcus...Klebsiella aerogenes, K. pneumoniae, Methylophilus methylotrophus, Moraxella spp., Mycobacterium aurum...Klebsiella spp., Legionella pneumophila, Methylophilus methylotrophus, Myxococcus virescens, M. xanthus, Proteus...eutrophus, Azotobacter vinelandii, Brevibacterium methylicum, Caulobacter crescentus, Desulfovibrio vulgaris...

...Table 2: Lab strains of E. coli Strain Natural resistance Primary Use Genotype ccdB Survival 2 T1R ...resistance to tetracycline Additionally, Table 2 provides a quick reference for some of the popular... mk+) Unmethylated DNA not degraded, cell still can methylate DNA   hsdS(rk-,mk-) Unmethylated DNA not...Consequence dam DNA adenine methylase mutation (GATC) Preparing unmethylated DNA, important when trying... XbaI) dcm DNA cytosine methylase mutation (CCWGG) Preparing unmethylated DNA, important when trying...pathway that cleaves methylated cytosine DNA Allows for uptake of foreign (methylated) DNA mrr, Δ(mcrC-mrr...that cleaves methylated adenine or cytosine DNA Allows for uptake of foreign (methylated) DNA recA, ...

...Figure 2: dCas9-Tet1 demethylates cytosines at promoters and enhancers. This targeted demethylation is associated...CRISPR-based approach for targeted DNA demethylation. Cell Discovery 2: . https://doi.org/10.1038/celldisc...pX330a dCas9-LSD1 for targeted removal of H3K4me1/2 and H3K9me2 histone modifications. Like the TALE-LSD1...fused a TALEN to the Tet1 demethylase catalytic domain, could mediate demethylation and induce transcription... fused a TAL effector to LSD1 histone demethylase to demethylate enhancer regions (Mendenhall et al., ...Tet1 demethylase Ronggui Hu’s lab has created pdCas9-Tet1-CD for targeted cytosine demethylation in mammalian... dCas9-DNMT3A methylates cytosines at promoters and enhancers. This targeted methylation is associated...

... the two DNA strands and releasing topoisomerase (2).  These days commercially available TOPO kits provide...Taq polymerase using your favorite PCR protocol.  2. Set Up TOPO Cloning Reaction: Mix together the PCR...PubMed PMID: 1658796. PubMed Central PMCID: PMC52876. 2. Novel approach to molecular cloning and polynucleotide...ability of complementary basepairs adenine (A) and thymine (T) to hybridize and form hydrogen bonds. This ...vectors or cloning arms with overhanging 3´ deoxythymidine (T) residues that are covalently linked to ...

...Carballosa, et al, (2015) eNeuro, March/April 2015, 2(2) e0001-15.2015. Pubmed. Resources: http://openspim.org...orthogonal-plane fluorescence optical sectioning (OPFOS) (2). Arne Voie, David Burns and Francis Spelman focused...in LSFM. In LSFM the laser light sheet, typically 2-6  microns, illuminates only one thin plane of the...(10).  He used a solution of benzyl alcohol and methyl salicylate which was later modified by others to...subpopulation of neurons are brightly labeled with GFP (e.g. Thy1-YFP-H mice).  However, we recently developed AAV...

...the addition of Cre recombinase (Figure 2). Figure 2: A knockout targeting vector designed to... through an experiment designed to knock out exon 2 of a given gene.   Figure 1: A knockout targeting...For recombination to occur in a cell, a minimum of 2 kb of sequence homology is required, but 6 to 14 kb...construct to your target cells. After recombination, exon 2 of the targeted gene will be removed from the chromosome...the GFP/NeoR cassette is inserted in place of exon 2. Thus, the targeted gene is disrupted (knocked out...recombination event will remove NeoR only and leave exon 2 floxed, as seen in line 4 of Figure 4. Since the loxP...genome, a negative selection marker like the HSV thymidine kinase (HSV-tk) is included just outside one of...

... constructed 2 versions of this reporter to target the K9 (pcDNA3-K9 histone methylation reporter) and... 525.7570 (2015): 533-537. PubMed PMID: 26352475. 2. Bygren, Lars O., et al. "Change in paternal grandmothers...in their DNA methylation patterns, either large increases or decreases. Hypermethylation of CpG islands...hybridization, methylation-sensitive restriction enzyme digestion, DNA adenine methyltransferase identification...linker is a methyllysine binding domain (chromodomain), which binds selectively to lysine-methylated peptides...the results. When methylation occurs at the histone-derived peptide, the methyllysine binding domain causes...to recognise lysine 27 methylation. When tested in living cells where methylation of these sites was controlled...

...Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme...PAM site. Uridine is subsequently converted to thymidine through base excision repair, creating a C to ...

...generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the...recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required for recombineering...generic lambda red recombineering experiment (Figure 2). In the following sections, key steps that differ...confirmation of recombinant clones.    Figure 2: Overview of using Lambda Red recombineering system...pairs of homology to the targeted insert site (Figure 2, top).  These primers are typically ~70 nucleotides...bacteria that has key MMR proteins knocked out or 2) specially design ssDNA oligos to avoid MMR: 1) E....will have more unintended changes to their genomes. 2) Designing ssDNA oligos that avoid MMR activation:...

...digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://doi.org/10.1089/hgtb.2013.131 Lock,... virus Once you have transfected your cells, wait 2–5 days and then collect the supernatant and/or producer...quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Human ..., R., & Kleinschmidt, J. (1999). Titration of AAV-2 particles via a novel capsid ELISA: Packaging of genomes... genomes can limit production of recombinant AAV-2. Gene Therapy, 6(7), Article 7. https://doi.org/10.1038...Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material. Human Gene Therapy, 21...rAAV from your supernatant, you can simply use polyethylene glycol (PEG). We add 25 mL of 40% PEG solution...